This invention relates to novel borrelidin derivatives, more specifically to novel borrelidin derivatives prepared by transforming the carboxyl group on the cyclopentane ring of borrelidin. Furthermore, the invention relates to pharmaceutical compositions containing such compounds and to the use of these compounds for preparing pharmaceutical compositions.
The novel compounds according to the invention show valuable biological efficiency, namely they have remarkable angiogenesis-inhibiting effect and antimetastatic action.
It is known that the angiogenesis is a phenomenon where blood-vessels are formed in the organism and a new vascular system is formed. The angiogenesis has very different forms depending on the growth and function of the endothelial cells, by all means it can be considered as a certain kind of cascade reactions. The angiogenesis takes place under normal physiological circumstances like a part of the evolution and reproduction processes in the case of the embryo, foetus, placenta, uterus and similar organs. It may be, however, a part even of a pathological process which accompanies the wound healing, infection as well as tumour growth, furthermore promote the formation of tumour metastases.
From the literature the clinical observation is known for a long time that the majority of cancer tumorous patients die due to metastases. This situation has been improved in the recent years by the radiation therapy and chemotherapy but the results attained are by no means reassuring.
Owing to the results attained while studying the pathobiological features of the malignant diseases, in the last years a new trend of antitumour drug research has developed. It can be attributed to this new tendency that nowadays the planning of novel active agents is directed, besides the research of active agents inhibiting the growth of tumorous tissues, even towards other pathobiological events (immortalisation, metastases, apoptosis, angiogenesis) responsible for maintaining the malignity. From these events special attention deserves the neovascularisation (formation of novel blood-vessels) which ensures continuous blood supply for the growing tumours, since in lack of same the tumorous cells are killed. According to the conclusions of tumour-biological examinations the progression of malignant diseases can be considered to be a function of angiogenesis, and the transition of the premalignant period into the invasive period, further the induction of the dormant state of the tumorous cell population in direction of proliferation can be brought into close connection with the formation of blood vessels.
Thus, the drug research of antitumorous agents is aimed today at planning and developing molecules having novel points of attack, by the aid of which the healing of cancer can be rendered more efficient than before.
Among these novel molecules priority is given to the group of antiangiogenetic agents which, by inhibiting the neovascularization and consequently the formation of metastases, may open a new period in the therapy of tumorous diseases.
Several compounds are known for having angiogenesis-inhibiting effect. From these compounds the following are enumerated as examples, without being exhaustive: angiostatic steroids [Folkman, J. et al., Science 221, 719 (1993)] like the cortisone which inhibits the function of the endothelial cells; the medroxyprogesterone acetate which inhibits the production of plasminogen-activator by the endothelial cells [Nicosia, R. F. and Ottinetti, A., Lab. Invest. 63, 115 (1990)]; the fumagillin which inhibits the formation of tubules [Ingber, D. et al., Nature 348, 555 (1990)]; a polysaccharide sulfate (SD-4152) which inhibits the migration and multiplication of the endothelial cells; and the retinoic acid responsible for the differentiation and transformation of the endothelial cells [Tsutomu Oikawa, Kekkan to Naihi 2, 470 (1992)]. However, these substances did not work as angiogenesis-inhibitors in the clinical practice: some of them due to a strong side-effect and others due to an insufficient target effect.
The first, even clinically effective angiogenesis-inhibitor was the xcex1-interferon [Bronty-Boye, D. and Zetter, B. E., Science 208, 516 (1980); Sidky, Y. A. and Borden, E. C., Cancer Res., 47, 5155 (1987)]. At present the clinical trials of several angiogenesis-inhibiting compounds with different chemical structures are under investigation; such compounds are for example the derivatives of fumagillin, e.g. the AGM-1470 [Kusaka, M. et al., Biophys. Res. Comm. 174, 1070 (1991)]; the 3-(2,4-dimethylpyrrol-5-yl)-indolin-2-one (SU-5416), U.S. Pat. No. 5,792,783; the 5-methylisoxazole-4-carboxylic-N-[4-(trifluoromethyl)-phenyl]-amide (leflunomide, SU-101), U.S. Pat. No. 5,610,173; the 2(R)-isobutyl-3(S)-dihydroxy-N-[2,2-dimethyl-1(S)xe2x80x94(N-methylcarbamoyl)-propyl]-succinamide (marimastat); the 3xcex2-[{3-[(4-aminobutyl)-amino]-propyl}-amino]-5xcex1-cholestane-7,24-diol-24-hydrogen sulfate (squalamine), U.S. Pat. No. 5,192,756; the ZD4190, an inhibitor of vascular endothelial growth factor etc.
Recently Japanese authors have described that the known borrelidin [chemically: 2xe2x80x2-(7-cyano-8,16-dihydroxy-9,11,13,15-tetramethyl-18-oxo-oxacyclooctadeca-4,6-dien-2-yl)-cyclopentane-1xe2x80x2-carboxylic acid], which is a macrolid antibiotic comprising a 18-membered ring [Keller-Schierlein, W., Experientia 22, 476 (1966); Helvetica Chim. Acta 50, 731 (1967); Anderson, B. F. et al., Aust. J. Chem. 42, 717 (1989)] has angiogenesis-inhibiting effect due to the property that it induces the apoptosis of the cells forming capillary tubules [Wakabayashi, T. et al., J. Antibiot. 50, 671 (1997)]. Furthermore, it has been proved that it is effective against cell lines WiDr of human colon cancer and PC-3 of human prostate cancer (published Japanese patent applications Nos. 8-173,167 and 9-227,549).
Furthermore, it is known that the borrelidin exerts antibacterial, antiviral, herbicidal and insecticidal effects and has a medium LD50 value (Glasby, J. S., Encyclopedia of Antibiotics, p. 145, J. Wiley (editor), 1979).
It is known from the literature and it is supported even by our own investigations that the efficiency of borrelidin is directed towards two tumourbiological events: the proliferation on the one hand and the capillary formation by the endothelial cells, that is the angiogenesis, on the other hand. Although there is a difference in respect of the sensitivity of the two cellular functions (about a five fold difference exists in favour of capillary formation), this selectivity is yet of smaller degree when we consider the inhibition of cell proliferation aimed at other cell kinds.
The invention aims at separating the two cell-biological effects by modifying the structure of borrelidin. More specifically, the invention aims at preparing, by transforming the carboxyl group on the cyclopentane ring of the borrelidin molecule, novel borrelidin derivatives which exert a much stronger effect on the capillary formation by the endothelial cells than on the cell proliferation. Namely, according to our hypothesis, in the clinical practice an angiogenesis-inhibiting active agent is needed which inhibits the cell proliferation only in higher doses. (Here it is mentioned that the selectivity of the known angiogenesis-inhibiting compounds prevails in the fact that they inhibit the proliferation of endothelial cells more definitely than the division of the other cells of the organism.)
During our investigations it has been surprisingly observed that the novel borrelidin derivatives of general formula (I) 
fully satisfy the above aims.
This recognition is surprising for a person skilled in the art because only a few borrelidin derivatives are known in the literature, i.e. its methyl ester and the diacetate of the methyl ester were prepared by Anderson, K. and Rickards, R. W. [Nature 206, 269 (1965)], further its benzyl ester and the bis-O-(4-nitrobenzoyl) derivative of the borrelidin methyl ester were described by Berger, J. et al. [Arch. Biochem. 22, 476 (1949)], and no any biological effect of these compounds was mentioned by the above authors. On the other hand, according to the literature only those borrelidin derivatives have angiogenesis inhibiting effect in which the carboxyl group located on the cyclopentane ring is not substituted. Such compounds are described for example in the published Japanese patent application JP 09227549-A (Kokai) which specifies compounds wherein a nitrile or carboxyl group is bonded to the carbon atom in position 7 of the borrelidin skeleton and a hydrogen atom or a lower alkyl group is bonded to the carbon atom in position 9.
Based on the above, the invention relates to compounds of general formula (I)xe2x80x94wherein
R stands for a group of the general formula xe2x80x94COOR1, xe2x80x94CONR2R3, xe2x80x94CONR4CONR4R5 or xe2x80x94CH2OR6, wherein
R1 stands for a C2-6 alkyl group; a C1-6 alkyl group substituted by hydroxyl, amino, di(C1-4 alkyl)-amino group or 5-8-membered saturated, nitrogen-containing heterocyclic group (which may comprise beside the nitrogen atom even an oxygen atom or one or two further nitrogen atoms) or by 5- or 6-membered, nitrogen-containing aromatic heterocyclic group (which may comprise beside the nitrogen atom even an oxygen atom or one or two further nitrogen atoms); or a C3-6 cycloalkyl group;
R2 and R3 are identical or different and stand independently from each other for hydrogen atom or a C1-6 alkyl group which optionally may be substituted by halogen atom, hydroxyl, amino, C2-5 alkoxycarbonyl, di(C1-4 alkyl)-amino group or 5-8-membered saturated, nitrogen-containing heterocyclic group (which may comprise beside the nitrogen atom even an oxygen atom or one or two further nitrogen atoms) or 5- or 6-membered aromatic homocyclic group or aromatic heterocyclic group containing an oxygen and/or nitrogen atom; a 5- or 6-membered cycloalkyl or a heteroaryl group;
R4 and R5 are identical or different and stand independently from each other for a hydrogen atom, a C1-6 alkyl group, a C3-6 cycloalkyl group or an optionally substituted phenyl group;
R6 stands for a hydrogen atom; a C1-5 alkyl, a C3-6 cycloalkyl or a C2-6 aliphatic acyl group which optionally may be substituted by halogen atom, amino, di(C1-4 alkyl)-amino or optionally substituted phenyl group; an optionally substituted carbamoyl group, an optionally substituted benzoyl group or a C1-4 alkylsulfonyl groupxe2x80x94
and their tautomers, solvates, the mixtures thereof and the acid-addition salts of all these compounds.
Here it is mentioned that in the drawing of the general formula (I) the letters (R) and (S) designate the absolute configuration of the corresponding carbon atoms.
In the enumeration of the meanings of the substituents of the compounds of general formula (I) the designation xe2x80x9calkyl groupxe2x80x9d relates both to straight or branched chain groups. Such groups are for example the ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1-ethyl-propyl, hexyl and isohexyl groups.
The cycloalkyl group can be cyclopropyl, cyclopentyl or cyclohexyl group.
The halogen atom can be chlorine or bromine atom.
In the meaning of R1, R2 and R3 the 5-8-membered saturated, nitrogen-containing heterocyclic group can be for example but not exclusively 1-pyrrolidinyl, 1-piperidinyl, hexa-hydro-1H-azepin-1-yl, octahydroazocin-1-yl, piperazinyl and morpholinyl group.
In the meaning of R2 and R3 the C2-5 alkoxycarbonyl group can be for example but not exclusively carbometoxymethyl, carbo-t-butyloxymethyl, carbomethoxyethyl and carbometoxypropyl group.
In the meaning of R2 and R3 the 5- or 6-membered aromatic homocyclic group can be for example but not exclusively phenyl or substituted phenyl group.
In the meaning of R1, R2 and R3 the designations xe2x80x9c5- or 6-membered aromatic heterocyclic group containing oxygen and/or nitrogenxe2x80x9d and xe2x80x9cheteroaryl groupxe2x80x9d, resp., relate for example but not exclusively to the following groups: furyl, pyrrolyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,5-triazinyl, 2-pyridyl, 3-pyridyl and 4-pyridyl groups.
In the meaning of R6 the designation xe2x80x9cC2-6 aliphatic acyl groupxe2x80x9d relates for example to acetyl, propionyl, butanoyl, isobutanoyl, sec-butanoyl, tert-butanoyl, n-caproyl or isocaproyl group. The term xe2x80x9cC1-4 alkylsulfonyl group relates for example to methanesulfonyl or ethanesulfonyl group. Furthermore, the term xe2x80x9coptionally substituted carbamoyl groupxe2x80x9d relates to carbamoyl, C1-6 alkylcarbamoyl, C3-6 cycloalkylcarbamoyl or carbamoyl groups substituted by C2-6 aliphatic acyl group, which optionally may be substituted by a halogen atom, like e.g. the chloroacetyl-carbamoyl group.
Under the term xe2x80x9csaltsxe2x80x9d formed with the compounds of general formula (I) the salts formed with physiologically acceptable inorganic and organic acids should be understood. Such acids suitable for salt-formation are e.g. the hydrochloric acid, the hydrobromic acid, the phosphoric acid or the sulfuric acid. For example formic acid, acetic acid, maleic acid, fumaric acid, succinic acid, lactic acid, citric acid or methanesulfonic acid can be used as organic acid.
An advantageous group of the compounds of general formula (I) according to the invention comprises compounds of general formula (I), wherein R stands for general formula xe2x80x94CONR2R3, wherein R2 and R3 stand for hydrogen atom or one of R2 and R3 stands for hydrogen atom and the other represents a C1-6 alkyl group substituted by a 5- or 6-membered aromatic heterocyclic group containing a nitrogen atom.
For preparing the compounds of general formula (I) the esterifying, amidating and reducing, resp., methods can be used which are generally known from the literature, e.g. from the Synthetic Organic Chemistry (Wagner, R. B. and Zook, H. D., Wiley, N.Y., 1956).
The compounds of general formula (I) can be prepared by adapting the above general methods, for example by using the following processes:
a) reaction of an acid chloride formed from borrelidin with a suitable alcohol or amine,
b) direct esterification or amidation of borrelidin in the presence of carbodiimide and a base,
c) transesterification of an ester, formed from borrelidin, with a suitable alcohol,
d) reaction of borrelidin methyl ester with a suitable amine,
e) formation of an active ester from borrelidin e.g. with N-hydroxybenztriazole, then reaction with a suitable alcohol or amine,
f) formation of a mixed anhydride from borrelidin e.g. with chloroformic acid ester, then reaction with a suitable amine,
g) reduction of a mixed anhydride formed from borrelidin with a metal hydride to an alcohol,
h) alkylation and acylation, resp., of an alcohol prepared from borrelidin.
We have found that the novel ester derivatives of borrelidin according to the invention can be most preferably prepared by reacting an active derivative, formed from borrelidin with N-hydroxybenztriazole in the presence of carbodiimide, with a suitable alcohol.
The reaction is carried out in inert solvents, most preferably in tetrahydrofuran. In the process dicyclohexylcarbodiimide (DCC) is used as carbodiimide and dimethylaminopyridine (DMAP) is used as a base. It is suitable to use an excess of 10 moles from the alcohol component. The reaction is carried out at a temperature between 0xc2x0 C. and 50xc2x0 C., preferably at 20xc2x0 C., under stirring during 1-8 hours, preferably during 3 hours.
The acid amide derivatives of borrelidin can be very preferably prepared for example with the mixed anhydride derivative formed with chloroformic acid ester. The reaction can be carried out in inert water-free solvents like e.g. tetrahydrofuran, dichloromethane, carbon tetrachloride. Triethyl amine, pyridine and dimethylaminopyridine can be used as acid-binding agent. From the amine to be coupled 1-10 moles can be used. The reaction is carried out by stirring at a temperature between xe2x88x9220xc2x0 C. and +20xc2x0 C., during 1-8 hours. In our most preferred process the mixed anhydride derivative is formed in water-free tetrahydrofuran at xe2x88x9220xc2x0 C., in the presence of triethyl amine, with isobutyl chloroformate, then the reaction is carried out with 5 moles of amine during 3 hours.
The alcohol derivative of borrelidin can be prepared most preferably from a suitable mixed anhydride derivative of borrelidin by reduction carried out with an aqueous complex metal hydride, preferably sodium borohydride, in tetrahydrofuran at xe2x88x9220xc2x0 C.
The alkylation and acylation, resp., of the alcohol derivative of borrelidin can be carried out in a way known per se.
It is evident for a person skilled in the art that when preparing compounds of general formula (I), which have starting compounds wherein certain substituents contain reactive group(s) not to be transformed in a given reaction, then this (these) group(s) can be protected in a manner known per se in the organic chemistry, and the protective group(s) are removed after the given reaction in such a way that other parts of the molecule should not undergo any undesired transformation. For protecting the said groups, usually employed protective groups known per se can be used. Such protective groups are known for example from the book xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d by Greene, T. W. and Wuts, P. (John Wiley and Sons, New York, 1991).
A part of the compounds of general formula (I) according to the invention contains a basic N atom which is suitable for forming salts. Such bases of general formula (I) can be transformed toxe2x80x94preferably pharmaceutically acceptablexe2x80x94acid-addition salts in a known way, for example by dissolving the base in a suitable organic solvent and adding the suitable acid or a solution of the acid, prepared with a suitable organic solvent. The thus-obtained salt is separated by filtration or by evaporating the solvent in vacuo and, if desired, it can be purified in a known way, for example by recrystallisation.
As mentioned above, the compounds of general formula (I) according to the invention have valuable biological efficiency, namely they show remarkable angiogenesis-inhibiting effect which is accompanied by a very favourable selectivity.
The angiogenesis-inhibiting effect of the compounds according to the invention has been determined by measuring the effect on the proliferation of and the capillary formation by the endothelial cells. The test methods are shown below.
Examination of Cell Proliferation
The endothelial cells ECV 304 (DSMZ No. ACC310) were propageted in an in vitro monolayer culture in a culture medium RPMI 1640 (SIGMA, USA) containing 10% fetal calf serum (Protein GMK, Gxc3x6dxc3x6llxc3x6, Hungary). The borrelidin and its novel derivatives of general formula (I) according to the invention were added in the exponential period of the culture for attaining the different end concentrations (0.1-100 xcexcg/ml). The growth of the cell culture was followed in a Fluoroscan Ascent FL apparatus on the basis of the change in the amount of the DNA, measured with the aid of the Hoechst 33342 stain.
Similar proliferation-inhibiting effect of borrelidin and the compounds according to the invention of general formula (I) on the endothelial proliferation could be observed even on a cell culture prepared from human umbilical cord (HUVEC).
Examination of Endothelial Capillary Formation
Basal membrane protein gel prepared from murine EHS tumor, which induces capillary formation, was put onto ECV 304 endothelial cells. The treatment with the borrelidin and the novel borrelidin derivatives of general formula (I) according to the invention was carried out in the same way as in case of the examination of cell proliferation. The extent by which the cells take part in the capillary formation was examined microscopically and by the aid of a morphometrical program, and the thus-obtained data were expressed in percent of the untreated control.
Results
The two methods proved to be suitable for demonstrating that the novel borrelidin derivatives according to the invention fulfil the requirement of selectively inhibiting the capillary formation. Namely, it has been stated that, relating to borrelidin, the cell-proliferation-inhibiting effect of the novel derivatives of general formula (I) definitely decreases whereas the capillary-formation-inhibiting effect changes only in a small degree or not at all. The relative degree of selectivity was determined for each compound by multiplying the ratios of the active agent concentrations that inhibit the cell proliferation in 50% and which inhibit the capillary formation. (The ratios were formed by dividing the suitable inhibiting concentration of the novel compound according to the invention by the suitable inhibiting concentration of borrelidin.) On the basis of the thus-calculated selectivity index the compound of Example 1 inhibits the capillary formation 60 times, the compound of Example 3 37 times, the compound of Example 2 7.5 times and the compound of Example 4 6 times better than the cell proliferation, in relation to the borrelidin.
The test data relating to the inhibition of formation of capillary tubules were confirmed when using the xe2x80x9cmicrovessel formationxe2x80x9d method [Parish et al., Cancer Res. 59, 3433 (1999)]. This method enabled us to study the neovascularization in a tissue culture formed from the artery of human placenta. We could state that the borrelidin derivatives according to the invention considerably inhibit the propagation of endothelial cells and even better the formation of tubules.
Based on our test results we could conclude to the fact that the novel borrelidin derivatives according to the invention act primarily on a cellular mechanism which is able to interrupt the capillarisation of endothelial cells and influences the proliferation of such cells only in a higher concentration. Our recognition that in the same endothelial cell culture the novel borrelidin derivatives according to the invention inhibit the tubule formation in a lower concentration than the proliferation of the endothelial cells, is not only novel but even surprising. Consequently, the selectivity does not appear in the dissimilar sensitivity of different cells but it can be observed between the intercellular connections, directing the capillary formation, and the cell proliferation.
Investigation into the Antitumorous Effect in Metastasis Model Systems
1. In a Lewis lung adenocarcinoma model [Holmgren et al., Nature Medicine 1, 149 (1995)] the borrelidin inhibited in a very small degree the propagation of metastatic nodules being formed after removing the primary tumour in lung. On the other hand, the compound according to Example 15 inhibited very considerably the increase of micrometastases not only with intraperitoneal but also with per os administration when adding one fifth of the toxic dose.
2. In a colon 38 spleen-liver model [Dong, Z. et al., J. Natl. Cancer Inst. 86, 913 (1994); Shaheen, R. M. et al., Cancer Research 89, 5412 (1999)] test system the metastasis-forming ability of mouse colon adenocarcinoma cells transplanted into the spleen considerably decreased after the subtoxic administration of the compound of Example 15.
The compounds according to the invention can be used for therapeutical purposes either alone or preferably in the form of pharmaceutical compositions. Such compositions fall under the scope of the present invention.
These pharmaceutical compositions contain a compound of general formula (I) in an amount necessary for exerting the desired effect, together with carriers, fillers, diluents and/or other pharmaceutical auxiliary materials known per se and generally used in the pharmaceutical industry.
For example water, alcohols, gelatine, lactose, saccharose, starch, pectin, magnesium stearate, stearic acid, talc, various animal or plant oils as well as glycols, such as propyleneglycol or polyethylenglycol, can be used as carriers, diluents and fillers mentioned above. As pharmaceutical auxiliary materials e.g. preservatives, antioxidants, different natural or synthetic emulsifying, dispersing or wetting agents, colouring agents, flavouring agents, buffering agents, disintegrating agents and other materials enhancing the biological utilization of the active agent can be used.
The pharmaceutical compositions according to the invention may be in the usual forms, such as oral preparations which can be prepared by using the above-mentioned pharmaceutical auxiliary agents. These oral compositions may be solid pharmaceutical forms such as tablets, capsules, powders, pills, dragxc3xa9es or granules, or liquid pharmaceutical forms such as syrups, solutions, emulsions or suspensions. The rectal preparations can be suppositories. The parenteral preparations, that are administered by avoiding the gastric system, may be for example injection or infusion solutions. Further, the pharmaceutical compositions according to the invention can be external preparations such as ointments, creams, water for compresses, eye-rinsing solutions, eyedrops etc.
Although the dose of the compounds according to the invention necessary for exerting the required pharmaceutical effect depends inter alia on the individual state and age of the patient and is finally determined by the doctor, for preventing and/or treating diseases, wherein the inhibition of angiogenesis appearing in connection with the disease is requested, a dose between about 0.5 mg and about 100 mg per 1 kg of body weight can be used. This dose can be administered daily in several portions, considering even the conditions of absorption.
The pharmaceutical compositions comprising compounds of general formula (I) according to the invention can be used beside surgical intervention and radiotherapy as adjuvants primarily for treating and preventing the increase of tumours and for limiting the formation of cancer metastases. Besides, they can be used for treating other diseases and states where the inhibition, control and/or regression of vascularisation exerts a favourable effect; here we mention as examples the arthritis, various ophthalmological cases (e.g. subretinalis neovascularisatio) as well as psoriasis.
Based on the above, the present invention also provides a method of treating angiogenic diseases in mammals; caused by excessive inappropriate angiogenesis, which comprises administering to a mammal in need of such treatment an effective dosage of a compound of general formula (I).
The compounds according to the invention and the process for their preparation are further illustrated by the following non-limiting examples.